Abstract Body (Do not enter title and authors here):
Background
β₁-adrenergic autoantibodies (β₁-AA), pathogenic autoantibodies targeting β₁-AR, drive adverse cardiac remodeling and elevate rehospitalization risk in HF. It has been established that β₁-AA induce both proportional imbalances and functional dysregulation of CD4⁺ T lymphocytes. However, the mechanisms underlying β₁-AA-induced CD4⁺ T cell dysfunction and its pathophysiological relationship with HF progression remain unelucidated.

Methods and Results
Peripheral blood was collected from heart failure (HF) patients and analyzed by flow cytometry, revealing a significant reduction in CD28 expression on CD4⁺ T cells along with increased granzyme secretion in HF patients. HF mouse model (passive transfer of heart failure patient derived β₁-AA, H-β₁-AA) exhibited enhanced cardiac infiltration of CD4⁺ T cells and immune dysregulation in peripheral blood. In vivo, H-β₁-AA stimulated CD4⁺ T cells were adoptively transferred into nude mice, resulting in cardiac dysfunction accompanied by massive CD4⁺ T cell infiltration into the heart. Cell coculture confirmed that H-β₁-AA stimulated CD4⁺ T cells induced myocardial injury in vitro. Moreover, CD28 downregulation was consistently observed in: splenic CD4⁺ T cells from β₁-AA passively immunized mice, cardiac-infiltrating CD4⁺ T cells in nude mice, and in vitro-stimulated CD4⁺ T cells by H-β₁-AA. Proteomic analysis of β₁-AA-stimulated CD4⁺ T cells revealed enhanced CD28 endocytosis. Further experiments employing spinning-disk confocal microscopy and related techniques demonstrated that β₁-AA stimulation induces CD28 internalization via macropinocytosis. Upon Ehd2 (a pivotal regulator of macropinocytosis) knockdown in CD4⁺ T cells, the heart failure phenotype induced by β₁-AA-CD4⁺ T cells in nude mice was reversed. β₁-AA-CD4⁺ T cells were then subjected to IP with anti-CD28 antibody, followed by mass spectrometry analysis, and validated, which identified increased GRP75 translocation to membrane and interaction with CD28 upon β₁-AA stimulation. The GRP75 inhibitors MKT-077 and Withanone reversed β₁-AA-induced macropinocytosis and CD28 loss in CD4⁺ T cells. Withanone also reversed the HF phenotype in nude mice receiving β₁-AA-CD4⁺ T cells.

Conclusion
Our study reveals for the first time that β₁-AA induce GRP75 membrane translocation, which mediates CD28 molecule internalization via macropinocytosis in CD4⁺ T cells, ultimately contributing to heart failure pathogenesis.